Rapid, accurate diagnostics for category A biological agents and other microbial pathogens, which could be converted into bioweapons, are essential in preparing for a possible bioterrorist attack. The surveillance for these agents and their associated diseases has to be carried out at the local and state level. This calls for a fast, unified, high-throughput assay that can be performed by minimally trained personnel in the current health care system and at low fixed cost. Furthermore, this diagnostic needs to be rapidly scalable in case of a local outbreak and it needs to be able to distinguish between imminent infections and harmless past exposure such as attenuated virus vaccination of an individual. These requirements exclude specialized, culture-based methods, as well as conventional serological assays, since these cannot distinguish between past exposure and ongoing infection (e.g. in the case of HIV-1 infection). Based on our previously published expertise, we propose to adopt high-throughput, quantitative real-time PCR to category biological agent surveillance. We will develop a multi-well panel of quantitative real-time RT-PCR and PCR primers, which can specifically and selectively quantify all suspected bioterrorist pathogens. Using a unified methodology (PCR) for all agents will (a) yield a faster local response time, (b) be more cost-effective, and (c) be more consistent, removing the variability, inherent in current culture and serology tests. We will apply this novel technology to homeland defense and integrate the superior technology into current operations at the Oklahoma City Health department, conduct training, and assess the validity of our approach. Our approach is based on the technical expertise of this NEW INVESTIGATOR and is of the limited scope appropriate to an R21 application. It utilizes the established capability in bacterial genomics at the University of Oklahoma Health Sciences Center and is greatly motivated by Oklahoma City's unfortunate experience with a large-scale terrorist attack.